Transfer RNAs (tRNAs) perform central functions in the cell and their activities depend on the presence of critical modified nucleotides in their structures. For this reason, cells devote numerous components (modification enzymes and their regulators) and significant energy to modify the bases and the riboses (RNA modifications). Two important modifications, uracil (U) to pseudouridine (?f?) isomerization at position 55 and substitution of guanine (G) by queuine (0) at position 34 of a tRNA, are the focus of this study. These reactions are catalyzed by pseudouridine 55 synthase (W55S) and tRNA-guanine transglycosylase (TGT), respectively. In this proposal, we will address issues related to the molecular recognition and mechanisms of these two modification reactions. Various alterations in bases and tRNA substrates are proposed. These modified substrates will be chemically synthesized in order to freeze or trap enzyme.tRNA intermediates at different stages along a reaction pathway. Conventional techniques for biochemical assays, such as electrophoretic mobility shift assay, chromatographic and spectroscopic methods, will be applied to characterize the freezing or trapping processes. As soon as stalled or trapped enzyme. tRNA complexes are obtained, crystallization will be carried out in order to obtain structural information of these complexes. Although under-modification of W at position 55 of a tRNA has not yet been directly correlated to disease, the deletions and mutations of the corresponding human pseudouridine synthase, dubbed dyskerin, which is strongly homologous to psi55S but involved in U to psi isomerization in ribosome RNA (rRNA), causes dykeratosis congenita (DKC), a rare inherited disorder. Patients with this disease have an age-dependent increase in risk for developing certain cancers, such as epithelial tumors of the skin and gastrointestinal tract. On the other hand, the extent of Q under-modification was correlated with morphological characterization of human leukemia, lymphoma, and lung tumors. The studies proposed here will reveal the basis for molecular recognition of tRNAs by these enzymes and probe the mechanisms of the modification reactions. The results will likely contribute to our understanding of the possible molecular causes of these diseases.